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Abstract：
BACKGROUND: Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease.
OBJECTIVE: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism.
METHODS: The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape.
RESULTS: Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 μM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay.
CONCLUSIONS: (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one.
OBJECTIVE: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism.
METHODS: The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape.
RESULTS: Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 μM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay.
CONCLUSIONS: (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7\'E,8S)-2\',4,8-trihydroxy-3-methoxy-2,4\'-epoxy-8,5\'-neolign-7\'-en-7-one.
摘要：
背景：中国胸已经在东亚用于治疗胆囊炎，传染性肝炎，黄疸和治疗肝脏问题。最近的证据为P.chinense在临床上用于治疗代谢性疾病提供了潜力。
目的：基于传统的使用和最近的证据，我们研究了具有调节功能的P.chinense成分对前蛋白转化酶枯草杆菌蛋白酶/kexin9型（PCSK9）和低密度脂蛋白受体（LDLR）表达的影响，以及最具活性物质对胆固醇摄取的影响，和与脂质代谢相关的基因。
方法：使用色谱方法从P.chinense的80％甲醇提取物的BuOH可溶性提取物中分离化合物，并通过解释光谱数据建立结构。实时定量PCR,进行蛋白质印迹分析以监测对PCSK9和LDLR表达的调节活性。还测试了PCSK9-LDLR结合相互作用。使用1,1-双十八烷基-3,3,3,3-四甲基吲哚卡布花青高氯酸盐（DiI）标记的LDL胆固醇测量肝细胞中的胆固醇吸收。此外,进行了LDLR的基因网络分析及其靶蛋白的反应，以发现与活性化合物对HepG2细胞的作用密切相关的基因。此外，我们通过String进行了蛋白质-蛋白质相互作用分析，并使用Cytoscape构建了复合靶网络。
结果：从P.chinense中表征了两种新的新木脂素和37种已知化合物。在分离的化合物中，(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新共轭-7'-en-7-酮(3)，发现penthorinA（4）和没食子酸甲酯（25）抑制PCSK9mRNA表达，IC50值为5.13，15.56和11.66μM，分别。然而,在PCSK9-LDLR相互作用试验中,所有分离的化合物均无活性.此外,一种二苯并恶卓类木酚素类似物,(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新良性-7'-en-7-酮（3）证明通过转录因子固醇调节元件-结合蛋白2（SREBP2）上调LDLRmRNA和蛋白质表达。此外，(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新良性-7'-en-7-酮（3）增加DiI-LDL测定中LDL-胆固醇的摄取。
结论：(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新良性-7'-en-7-酮（3）似乎通过下调PCSK9和激活LDLR来增加潜在的胆固醇摄取在肝细胞中。此外，SREBP2被发现在PCSK9和LDLR的调节中起重要作用（7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新共轭-7'-烯-7-酮。
目的：基于传统的使用和最近的证据，我们研究了具有调节功能的P.chinense成分对前蛋白转化酶枯草杆菌蛋白酶/kexin9型（PCSK9）和低密度脂蛋白受体（LDLR）表达的影响，以及最具活性物质对胆固醇摄取的影响，和与脂质代谢相关的基因。
方法：使用色谱方法从P.chinense的80％甲醇提取物的BuOH可溶性提取物中分离化合物，并通过解释光谱数据建立结构。实时定量PCR,进行蛋白质印迹分析以监测对PCSK9和LDLR表达的调节活性。还测试了PCSK9-LDLR结合相互作用。使用1,1-双十八烷基-3,3,3,3-四甲基吲哚卡布花青高氯酸盐（DiI）标记的LDL胆固醇测量肝细胞中的胆固醇吸收。此外,进行了LDLR的基因网络分析及其靶蛋白的反应，以发现与活性化合物对HepG2细胞的作用密切相关的基因。此外，我们通过String进行了蛋白质-蛋白质相互作用分析，并使用Cytoscape构建了复合靶网络。
结果：从P.chinense中表征了两种新的新木脂素和37种已知化合物。在分离的化合物中，(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新共轭-7'-en-7-酮(3)，发现penthorinA（4）和没食子酸甲酯（25）抑制PCSK9mRNA表达，IC50值为5.13，15.56和11.66μM，分别。然而,在PCSK9-LDLR相互作用试验中,所有分离的化合物均无活性.此外,一种二苯并恶卓类木酚素类似物,(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新良性-7'-en-7-酮（3）证明通过转录因子固醇调节元件-结合蛋白2（SREBP2）上调LDLRmRNA和蛋白质表达。此外，(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新良性-7'-en-7-酮（3）增加DiI-LDL测定中LDL-胆固醇的摄取。
结论：(7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新良性-7'-en-7-酮（3）似乎通过下调PCSK9和激活LDLR来增加潜在的胆固醇摄取在肝细胞中。此外，SREBP2被发现在PCSK9和LDLR的调节中起重要作用（7\'E，8S)-2\',4,8-三羟基-3-甲氧基-2,4'-环氧-8,5'-新共轭-7'-烯-7-酮。
